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The biological clocks of the circadian timing system coordinate cellular and physiological processes and synchronize them with daily cycles. While the central clock in the suprachiasmatic nucleus (SCN) is mainly synchronized by the light/dark cycles, the peripheral clocks react to other stimuli, including the feeding/fasting state, nutrients, sleep-wake cycles, and physical activity. During the disruption of circadian rhythms due to genetic mutations or social and occupational obligations, incorrect arrangement between the internal clock system and environmental rhythms leads to the development of obesity. Desynchronization between the central and peripheral clocks by altered timing of food intake and diet composition leads to uncoupling of the peripheral clocks from the central pacemaker and to the development of metabolic disorders. The strong coupling of the SCN to the light-dark cycle creates a situation of misalignment when food is ingested during the "wrong" time of day. Food-anticipatory activity is mediated by a self-sustained circadian timing, and its principal component is a food-entrainable oscillator. Modifying the time of feeding alone greatly affects body weight, whereas ketogenic diet (KD) influences circadian biology, through the modulation of clock gene expression. Night-eating behavior is one of the causes of circadian disruption, and night eaters have compulsive and uncontrolled eating with severe obesity. By contrast, time-restricted eating (TRE) restores circadian rhythms through maintaining an appropriate daily rhythm of the eating-fasting cycle. The hypothalamus has a crucial role in the regulation of energy balance rather than food intake. While circadian locomotor output cycles kaput (CLOCK) expression levels increase with high-fat diet-induced obesity, peroxisome proliferator-activated receptor-alpha (PPARα) increases the transcriptional level of brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like 1 (BMAL1) in obese subjects. In this context, effective timing of chronotherapies aiming to correct SCN-driven rhythms depends on an accurate assessment of the SCN phase. In fact, in a multi-oscillator system, local rhythmicity and its disruption reflects the disruption of either local clocks or central clocks, thus imposing rhythmicity on those local tissues, whereas misalignment of peripheral oscillators is due to exosome-based intercellular communication.Consequently, disruption of clock genes results in dyslipidemia, insulin resistance, and obesity, while light exposure during the daytime, food intake during the daytime, and sleeping during the biological night promote circadian alignment between the central and peripheral clocks. Thus, shift work is associated with an increased risk of obesity, diabetes, and cardiovascular diseases because of unusual eating times as well as unusual light exposure and disruption of the circadian rhythm.

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The biological clock of the suprachiasmatic nucleus (SCN) orchestrates circadian (approximately daily) rhythms of behaviour and physiology that underpin health. SCN cell-autonomous time-keeping revolves around a transcriptional/translational feedback loop (TTFL) within which PERIOD (PER1,2) and CRYPTOCHROME (CRY1,2) proteins heterodimerise and suppress trans-activation of their encoding genes (Per1,2; Cry1,2). To explore its contribution to SCN time-keeping, we used adeno-associated virus-mediated translational switching to express PER2 (tsPER2) in organotypic SCN slices carrying bioluminescent TTFL circadian reporters. Translational switching requires provision of the non-canonical amino acid, alkyne lysine (AlkK), for protein expression. Correspondingly, AlkK, but not vehicle, induced constitutive expression of tsPER2 in SCN neurons and reversibly and dose-dependently suppressed pPer1-driven transcription in PER-deficient (Per1,2-null) SCN, illustrating the potency of PER2 in negative regulation within the TTFL. Constitutive expression of tsPER2, however, failed to initiate circadian oscillations in arrhythmic PER-deficient SCN. In rhythmic, PER-competent SCN, AlkK dose-dependently reduced the amplitude of PER2-reported oscillations as inhibition by tsPER2 progressively damped the TTFL. tsPER2 also dose-dependently lengthened the period of the SCN TTFL and neuronal calcium rhythms. Following wash-out of AlkK to remove tsPER2, the SCN regained TTFL amplitude and period. Furthermore, SCN retained their pre-washout phase: the removal of tsPER2 did not phase-shift the TTFL. Given that constitutive tsCRY1 can regulate TTFL amplitude and period, but also reset TTFL phase and initiate rhythms in CRY-deficient SCN, these results reveal overlapping and distinct properties of PER2 and CRY1 within the SCN, and emphasise the utility of translational switching to explore the functions of circadian proteins.

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BACKGROUND: The natural light environment is far more complex than that experienced by animals under laboratory conditions. As a burrowing species, wild mice are able to self-modulate their light exposure, a concept known as light environment sampling behaviour. By contrast, under laboratory conditions mice have little opportunity to exhibit this behaviour. To address this issue, here we introduce a simple nestbox paradigm to allow mice to self-modulate their light environment. Dark nestboxes fitted with passive infrared sensors were used to monitor locomotor activity, circadian entrainment, decision making and light environment sampling behaviour. RESULTS: Under these conditions, mice significantly reduce their light exposure to an average of just 0.8 h across a 24 h period. In addition, mice show a distinct pattern of light environment sampling behaviour, with peaks at dawn and dusk under a ramped light dark cycle. Furthermore, we show that the timing of light environment sampling behaviour depends upon endogenous circadian rhythms and is abolished in mice lacking a circadian clock, indicating a feedback loop between light, the circadian clock and behaviour. CONCLUSIONS: Our results highlight the important role of behaviour in modifying the light signals available for circadian entrainment under natural conditions.

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The biological rhythms generated by the endogenous circadian clocks across the tree of life regulate numerous behavioural, metabolic and physiological processes. Although evidence from various studies in Drosophila melanogaster indicates the importance of the core circadian clock genes in the intricate interplay between the circadian clock and metabolism, little is known about the contribution of the circadian photoreceptor/s in this process. The deep brain circadian photoreceptor CRYPTOCHROME (CRY) is essential for resetting the clock in response to light and is also highly expressed in metabolically active tissues in Drosophila. In this study, we sought to explore the possible roles played by CRY in triglyceride metabolism. We observed that the cry mutant (cry01) flies exhibited increased starvation resistance and triglyceride levels under both 12-hour (h) light:12 h dark cycle (LD) and under constant light (LL) compared to the control w1118 flies. We also observed that cry01 flies had significantly increased food intake, glycogen concentrations and life span under LD. In addition, cryptochrome seemed to affect triglyceride levels in adult flies in response to calorie-restricted and high-fat diets. These results suggest a role for the circadian photoreceptor CRY in triglyceride metabolism in Drosophila.

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BACKGROUND: Numerous insect species undertake long-distance migrations on an enormous scale, with great implications for ecosystems. Given that take-off is the point where it all starts, whether and how the external light and internal circadian rhythm are involved in regulating the take-off behaviour remains largely unknown. Herein, we explore this issue in a migratory pest, Cnaphalocrocis medinalis, via behavioural observations and RNAi experiments. RESULTS: The results showed that C. medinalis moths took off under conditions where the light intensity gradually weakened to 0.1 lx during the afternoon or evening, and the take-off proportions under full spectrum or blue light were significantly higher than that under red and green light. The ultraviolet-A/blue light-sensitive type 1 cryptochrome gene (Cmedcry1) was significantly higher in take-off moths than that of non-take-off moths. In contrast, the expression of the light-insensitive CRY2 (Cmedcry2) and circadian genes (Cmedtim and Cmedper) showed no significant differences. After silencing Cmedcry1, the take-off proportion significantly decreased. Thus, Cmedcry1 is involved in the decrease in light intensity induced take-off behaviour in C. medinalis. CONCLUSIONS: This study can help further explain the molecular mechanisms behind insect migration, especially light perception and signal transmission during take-off phases.

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In the field, plants face constantly changing light conditions caused by both atmospheric effects and neighbouring vegetation. This interplay creates a complex, fluctuating light environment within plant canopies. Shade-intolerant species rely on light cues from competitors to trigger shade avoidance responses, ensuring access to light for photosynthesis. While research often uses controlled growth chambers with steady light to study shade avoidance responses, the influence of light fluctuations in real-world settings remains unclear. This review examines the dynamic light environments found in woodlands, grasslands, and crops. We explore how plants respond to some fluctuations but not others, analyse the potential reasons for these differences, and discuss the possible molecular mechanisms regulating this sensitivity. We propose that studying shade avoidance responses under fluctuating light conditions offers a valuable tool to explore the intricate regulatory network behind them.

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The magnetic compass sense of migratory songbirds is thought to derive from magnetically sensitive photochemical reactions in cryptochromes located in photoreceptor cells in the birds' retinas. More specifically, transient radical pairs formed by light-activation of these proteins have been proposed to account for the birds' ability to orient themselves using the Earth's magnetic field and for the observation that radiofrequency magnetic fields, superimposed on the Earth's magnetic field, can disrupt this ability. Here, by means of spin dynamics simulations, we show that it may be possible for the birds to orient in a monochromatic radiofrequency field in the absence of the Earth's magnetic field. If such a behavioural test were successful, it would provide powerful additional evidence for a radical pair mechanism of avian magnetoreception.

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Cryptochrome (Cry) in some species could act as a quantum senser to detect the inclination angle of geomagnetic field, the function of which attributes the magnetic sensitivity of spins of unpaired electrons in radical pair (RP) in CRY generated by blue light irradiation. However, the effect of blue light on the structure and molecular behavior of Cry has not been well investigated. We conducted the size exclusion chromatography (SEC) and small-angle X-ray scattering (SAXS) analyses to inspect the molecular structure and behavior of cryptochrome 4a (ErCry4a) from European robin, a representative magnetosensory animal. The results indicated that ErCry4a could form flat-shape oligomers. Moreover, blue light irradiation induced the contraction of the ErCry4a molecule at the monomer scale and simultaneously accelerated the two-dimensional oligomerization of ErCry4a. This oligomerization may enhance the regularity of the two-dimensional arrangement of ErCry4a molecules, providing a positive effect for detecting the inclination angle.

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Understanding how plants respond to temperature is relevant for agriculture in a warming world. Responses to temperature of the shoot have been characterized more fully than those of the root. Previous work on thermomorphogenesis in roots established that for Arabidopsis thaliana (Columbia) seedlings grown continuously at a given temperature, the root meristem produces cells at the same rate at 15 as at 25ºC and the root's growth zone is the same length. To uncover the pathway(s) underlying this constancy, we screened 34 A. thaliana genotypes for parameters related to growth and division. No line failed to respond to temperature. Behavior was little affected by mutations in phytochrome or other genes that underly thermomorphogenesis in shoots. However, a mutant in cryptochrome2 was disrupted substantially in both cell division and elongation, specifically at 15ºC. Among the 34 lines, cell production rate varied extensively and was associated only weakly with root growth rate; in contrast, parameters relating to elongation were stable. Our data are consistent with models of root growth that invoke cell non-autonomous regulation for establishing boundaries between meristem, elongation zone, and mature zone.

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Many environmental features are cyclic, with predictable changes across the day, seasons and latitudes. Additionally, anthropogenic, artificial-light-induced changes in photoperiod or shiftwork-driven novel light/dark cycles also occur. Endogenous timekeepers or circadian clocks help organisms cope with such changes. The remarkable plasticity of clocks is evident in the waveforms of behavioural and molecular rhythms they govern. Despite detailed mechanistic insights into the functioning of the circadian clock, practical means to manipulate activity waveform are lacking. Previous studies using a nocturnal rodent model showed that novel light regimes caused locomotor activity to bifurcate such that mice showed two bouts of activity restricted to the dimly lit phases. Here, we explore the generalizability of these findings and leverage the genetic toolkit of Drosophila melanogaster to obtain mechanistic insights into this unique phenomenon. We find that dim scotopic illumination of specific durations induces circadian photoreceptor CRYPTOCHROME-dependent activity bifurcation in male flies. We show circadian reorganization of the pacemaker circuit, wherein the 'evening' neurons regulate the timing of both bouts of activity under novel light regimes. Our findings indicate that such environmental regimes can be exploited to design light cycles, which can ease the circadian waveform into synchronizing with challenging conditions.

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Most insects enter diapause, a state of physiological dormancy crucial for enduring harsh seasons, with photoperiod serving as the primary cue for its induction, ensuring proper seasonal timing of the process. Although the involvement of the circadian clock in the photoperiodic time measurement has been demonstrated through knockdown or knockout of clock genes, the involvement of clock gene cryptochrome 1 (cry1), which functions as a photoreceptor implicated in photoentrainment of the circadian clock across various insect species, remains unclear. In bivoltine strains of the silkworm, Bombyx mori, embryonic diapause is maternally controlled and affected by environmental conditions experienced by mother moths during embryonic and larval stages. Previous research highlighted the role of core clock genes, including period (per), timeless (tim), Clock (Clk) and cycle (cyc), in photoperiodic diapause induction in B. mori. In this study, we focused on the involvement of cry1 gene in B. mori photoperiodism. Phylogenetic analysis and conserved domain identification confirmed the presence of both Drosophila-type cry (cry1) and mammalian-type cry (cry2) genes in the B. mori genome, akin to other lepidopterans. Temporal expression analysis revealed higher cry1 gene expression during the photophase and lower expression during the scotophase, with knockouts of core clock genes (per, tim, Clk and cyc) disrupting this temporal expression pattern. Using CRISPR/Cas9-mediated genome editing, we established a cry1 knockout strain in p50T, a bivoltine strain exhibiting clear photoperiodism during both embryonic and larval stages. Although the wild-type strain displayed circadian rhythm in eclosion under continuous darkness, the cry1 knockout strain exhibited arrhythmic eclosion, implicating B. mori cry1 in the circadian clock feedback loop governing behavior rhythms. Females of the cry1 knockout strain failed to control photoperiodic diapause induction during both embryonic and larval stages, mirroring the diapause phenotype of the wild-type individuals reared under constant darkness, indicating that B. mori CRY1 contributes to photoperiodic time measurement as a photoreceptor. Furthermore, photoperiodic diapause induction during the larval stage was abolished in a cry1/tim double-knockout strain, suggesting that photic information received by CRY1 is relayed to the circadian clock. Overall, this study represents the first evidence of cry1 involvement in insect photoperiodism, specifically in diapause induction.

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The Earth's magnetic field can provide reliable directional information, allowing migrating animals to orient themselves using a magnetic compass or estimate their position relative to a target using map-based orientation. Here we show for the first time that young, inexperienced herring (Clupea harengus, Ch) have a magnetic compass when they migrate hundreds of kilometres to their feeding grounds. In birds, such as the European robin (Erithacus rubecula), radical pair-based magnetoreception involving cryptochrome 4 (ErCRY4) was demonstrated; the molecular basis of magnetoreception in fish is still elusive. We show that cry4 expression in the eye of herring is upregulated during the migratory season, but not before, indicating a possible use for migration. The amino acid structure of herring ChCRY4 shows four tryptophans and a flavin adenine dinucleotide-binding site, a prerequisite for a magnetic receptor. Using homology modelling, we successfully reconstructed ChCRY4 of herring, DrCRY4 of zebrafish (Danio rerio) and StCRY4 of brown trout (Salmo trutta) and showed that ChCRY4, DrCRY4 and ErCRY4a, but not StCRY4, exhibit very comparable dynamic behaviour. The electron transfer could take place in ChCRY4 in a similar way to ErCRY4a. The combined behavioural, transcriptomic and simulation experiments provide evidence that CRY4 could act as a magnetoreceptor in Atlantic herring.

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The effects of high-intensity blue light (HIBL, 500/1000 µmol m-2s-1, 450 nm) on Solanum lycopersicum mutants with high pigment (hp) and low pigment (lp) levels and cryptochrome 1 (cry1) deficiency on photosynthesis, chlorophylls, phenols, anthocyanins, nonenzymatic antioxidant activity, carotenoid composition, and the expression of light-dependent genes were investigated. The plants, grown under white light for 42 days, were exposed to HIBL for 72 h. The hp mutant quickly adapted to 500 µmol m-2s-1 HIBL, exhibiting enhanced photosynthesis, increased anthocyanin and carotenoids (beta-carotene, zeaxanthin), and increased expression of key genes involved in pigment biosynthesis (PSY1, PAL1, CHS, ANS) and PSII proteins along with an increase in nonenzymatic antioxidant activity. At 1000 µmol m-2s-1 HIBL, the lp mutant showed the highest photosynthetic activity, enhanced expression of genes associated with PSII external proteins (psbO, psbP, psbQ), and increased in neoxanthin content. This mutant demonstrated greater resistance at the higher HIBL, demonstrating increased stomatal conductance and photosynthesis rate. The cry1 mutant exhibited the highest non-photochemical quenching (NPQ) but had the lowest pigment contents and decreased photosynthetic rate and PSII activity, highlighting the critical role of CRY1 in adaptation to HIBL. The hp and lp mutants use distinct adaptation strategies, which are significantly hindered by the cry1 mutation. The pigment content appears to be crucial for adaptation at moderate HIBL doses, while CRY1 content and stomatal activity become more critical at higher doses.

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Plants growing in dense vegetation stands need to flexibly position their photosynthetic organs to ensure optimal light capture in a competitive environment. They do so through a suite of developmental responses referred to as the shade avoidance syndrome. Belowground, root development is also adjusted in response to aboveground neighbour proximity. Canopies are dynamic and complex environments with heterogenous light cues in the far-red, red, blue and UV spectrum, which can be perceived with photoreceptors by spatially separated plant tissues. Molecular regulation of plant architecture adjustment via PHYTOCHROME-INTERACTING FACTOR (PIF) transcription factors and growth-related hormones such as auxin, gibberellic acid, brassinosteroids and abscisic acid were historically studied without much attention to spatial or tissue-specific context. Recent developments and technologies have, however, sparked strong interest in spatially explicit understanding of shade avoidance regulation. Other environmental factors such as temperature and nutrient availability interact with the molecular shade avoidance regulation network, often depending on the spatial location of the signals, and the responding organs. Here, we aim to review recent advances in how plants respond to heterogenous light cues and integrate these with other environmental signals.

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Marine fish migrate long distances up to hundreds or even thousands of kilometers for various reasons that include seasonal dependencies, feeding, or reproduction. The ability to perceive the geomagnetic field, called magnetoreception, is one of the many mechanisms allowing some fish to navigate reliably in the aquatic realm. While it is believed that the photoreceptor protein cryptochrome 4 (Cry4) is the key component for the radical pair-based magnetoreception mechanism in night migratory songbirds, the Cry4 mechanism in fish is still largely unexplored. The present study aims to investigate properties of the fish Cry4 protein in order to understand the potential involvement in a radical pair-based magnetoreception. Specifically, a computationally reconstructed atomistic model of Cry4 from the Atlantic herring (Clupea harengus) was studied employing classical molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) methods to investigate internal electron transfers and the radical pair formation. The QM/MM simulations reveal that electron transfers occur similarly to those found experimentally and computationally in Cry4 from European robin (Erithacus rubecula). It is therefore plausible that the investigated Atlantic herring Cry4 has the physical and chemical properties to form radical pairs that in turn could provide fish with a radical pair-based magnetic field compass sensor.

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Almost every facet of our behavior and physiology varies predictably over the course of day and night, anticipating and adapting us to their associated opportunities and challenges. These rhythms are driven by endogenous biological clocks that, when deprived of environmental cues, can continue to oscillate within a period of approximately 1 day, hence circa-dian. Normally, retinal signals synchronize them to the cycle of light and darkness, but disruption of circadian organization, a common feature of modern lifestyles, carries considerable costs to health. Circadian timekeeping pivots around a cell-autonomous molecular clock, widely expressed across tissues. These cellular timers are in turn synchronized by the principal circadian clock of the brain: the hypothalamic suprachiasmatic nucleus (SCN). Intercellular signals make the SCN network a very powerful pacemaker. Previously, neurons were considered the sole SCN timekeepers, with glial cells playing supportive roles. New discoveries have revealed, however, that astrocytes are active partners in SCN network timekeeping, with their cell-autonomous clock regulating extracellular glutamate and GABA concentrations to control circadian cycles of SCN neuronal activity. Here, we introduce circadian timekeeping at the cellular and SCN network levels before focusing on the contributions of astrocytes and their mutual interaction with neurons in circadian control in the brain.

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Photic entrainment is an essential function of the circadian clock, which enables organisms to set the appropriate timing of daily behavioral and physiological events. Recent studies have shown that the mechanisms of the circadian clock and photic entrainment vary among insect species. This study aimed to elucidate the circadian photoreceptors necessary for photic entrainment in firebrats Thermobia domestica, one of the most primitive apterygote insects. A homology search of publicly available RNA sequence (RNA-seq) data from T. domestica exhibited a cryptochrome 2 (cry2) gene and three opsin genes, opsin long wavelength 1 (opLW1), opLW2, and opUV, as candidate circadian photoreceptors. We examined the possible involvement of these genes in photic entrainment of firebrat locomotor rhythms. Firebrats had the highest entrainability to the light-dark cycle of green light. Treatment with dsRNA of the candidate genes strongly downregulated the respective targeted genes, and in the case of opsin genes, other untargeted genes were occasionally downregulated to various degrees. Under constant light, most control firebrats became arrhythmic, whereas a fraction of those treated with double RNAi of the two opLWs remained rhythmic. Behavioral experiments revealed that the transient cycles necessary for re-entrainment to shifted light cycles were lengthened when opLW2 expression was reduced. These results suggest that opLW2 is involved in the photic entrainment of circadian rhythm in firebrats.

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Cryptochromes are widely dispersed flavoprotein photoreceptors that regulate numerous developmental responses to light in plants, as well as to stress and entrainment of the circadian clock in animals and humans. All cryptochromes are closely related to an ancient family of light-absorbing flavoenzymes known as photolyases, which use light as an energy source for DNA repair but themselves have no light sensing role. Here we review the means by which plant cryptochromes acquired a light sensing function. This transition involved subtle changes within the flavin binding pocket which gave rise to a visual photocycle consisting of light-inducible and dark-reversible flavin redox state transitions. In this photocycle, light first triggers flavin reduction from an initial dark-adapted resting state (FADox). The reduced state is the biologically active or 'lit' state, correlating with biological activity. Subsequently, the photoreduced flavin reoxidises back to the dark adapted or 'resting' state. Because the rate of reoxidation determines the lifetime of the signaling state, it significantly modulates biological activity. As a consequence of this redox photocycle Crys respond to both the wavelength and the intensity of light, but are in addition regulated by factors such as temperature, oxygen concentration, and cellular metabolites that alter rates of flavin reoxidation even independently of light. Mechanistically, flavin reduction is correlated with conformational change in the protein, which is thought to mediate biological activity through interaction with biological signaling partners. In addition, a second, entirely independent signaling mechanism arises from the cryptochrome photocycle in the form of reactive oxygen species (ROS). These are synthesized during flavin reoxidation, are known mediators of biotic and abiotic stress responses, and have been linked to Cry biological activity in plants and animals. Additional special properties arising from the cryptochrome photocycle include responsivity to electromagnetic fields and their applications in optogenetics. Finally, innovations in methodology such as the use of Nitrogen Vacancy (NV) diamond centers to follow cryptochrome magnetic field sensitivity in vivo are discussed, as well as the potential for a whole new technology of 'magneto-genetics' for future applications in synthetic biology and medicine.

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Molecular and physiological determinants of the timing of reproductive events, including the pre-ovulatory LH surge and seasonal fluctuations in fertility, are incompletely understood. We used the Cryptochrome 1-deficient duper mutant to examine the role of this core circadian clock gene in Syrian hamsters. We find that the phase of the LH surge and its stability upon shifts of the light: dark cycle are altered in duper mutants. The intensity of immunoreactive PER1 in GnRH cells of the preoptic area peaks earlier in the day in duper than wild type hamsters. We note that GnRH fibers coursing through the suprachiasmatic nucleus (SCN) contact vasopressin- and VIP-immunoreactive cells, suggesting a possible locus of circadian control of the LH surge. Unlike wild types, duper hamsters do not regress their gonads within 8 weeks of constant darkness, despite evidence of melatonin secretion during the subjective night. In light of the finding that the duper allele is a stop codon in Cryptochrome 1, our results suggest important neuroendocrine functions of this core circadian clock gene.

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In this study, we investigate the intricate regulatory mechanisms underlying the circadian clock in Drosophila, focusing on the light-induced conformational changes in the cryptochrome (DmCry). Upon light exposure, DmCry undergoes conformational changes that prompt its binding to Timeless and Jetlag proteins, initiating a cascade crucial for the starting of a new circadian cycle. DmCry is subsequently degraded, contributing to the desensitization of the resetting mechanism. The transient and short-lived nature of DmCry protein-protein interactions (PPIs), leading to DmCry degradation within an hour of light exposure, presents a challenge for comprehensive exploration. To address this, we employed proximity-dependent biotinylation techniques, combining engineered BioID (TurboID) and APEX (APEX2) enzymes with mass spectrometry. This approach enabled the identification of the in vitro DmCry interactome in Drosophila S2 cells, uncovering several novel PPIs associated with DmCry. Validation of these interactions through a novel co-immunoprecipitation technique enhances the reliability of our findings. Importantly, our study suggests the potential of this method to reveal additional circadian clock- or magnetic field-dependent PPIs involving DmCry. This exploration of the DmCry interactome not only advances our understanding of circadian clock regulation but also establishes a versatile framework for future investigations into light- and time-dependent protein interactions in Drosophila.